A lung adenocarcinoma marker and application thereof in preparation of lung adenocarcinoma screening kit or treatment monitoring kit

Abstract

The application provides a lung adenocarcinoma marker circ_0006949 and application thereof in preparation of a lung adenocarcinoma screening kit or a treatment monitoring kit. The circ_0006949 in a sputum sample of a lung adenocarcinoma patient is significantly higher than that in a normal person, and the application first finds that the circ_0006949 is a biomarker for early diagnosis of lung adenocarcinoma; the sputum sample is non-invasive, and the detection price is low, so that the application is suitable for popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of lung adenocarcinoma screening and diagnosis, specifically relating to a lung adenocarcinoma screening biomarker and its diagnostic and treatment monitoring kit. Background Technology

[0002] Lung cancer is mainly divided into small cell lung cancer (14% of cases) and non-small cell lung cancer (NSCLC) (82% of cases) for treatment. NSCLC is a highly heterogeneous population, mainly divided into lung adenocarcinoma and lung squamous cell carcinoma. In recent years, with the widespread use of targeted and immunotherapies, such as angiogenesis inhibitors, epidermal growth factor receptor inhibitors, and programmed death ligand 1 and programmed cell death protein 1 inhibitors, the treatment of NSCLC has greatly improved. However, the mortality rate of NSCLC remains the highest among all cancers, mainly because the early symptoms of lung cancer are not obvious, most patients already have signs of metastasis when symptoms appear, and existing treatments are only effective for some patients, all of which are the main reasons for the high mortality rate. More than half (55%) of stage I or II NSCLC patients receive surgical treatment; in contrast, only 21% of stage III NSCLC patients receive surgical treatment, while the majority (61%) receive chemotherapy and / or radiation therapy. Therefore, early diagnosis and treatment are effective measures to reduce the mortality rate of NSCLC.

[0003] Currently, low-dose spiral CT (LDCT) is the primary method for screening early-stage lung cancer, significantly improving survival rates for lung cancer patients. However, high false-positive rates and radiation exposure are its drawbacks. Pathological diagnosis is the gold standard for NSCLC diagnosis, but the procedure of obtaining tissue biopsies has certain limitations and risks, and in many cases, tissue is not readily available. The effectiveness of traditional biomarkers is largely limited by the spatial and temporal heterogeneity of tumors, and the sensitivity and / or specificity of blood tumor markers such as carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and soluble fragment of cytokeratin 19 (Cyfra21-1) are not satisfactory.

[0004] Circular RNA (circRNA) is a single-stranded, covalently closed RNA molecule produced from pre-mRNA through a process called backsplicing. Unlike traditional linear RNA, circRNA is highly stable and can be found in exosomes, cell-free saliva, and plasma. circRNA may be involved in epithelial-mesenchymal transition, microRNA (miRNA) inhibition, and tumorigenesis. Furthermore, circRNA expression can be tissue-specific, and studies have shown that some non-coding circRNAs can be translated into proteins. With the development of high-throughput sequencing technologies and new bioinformatics algorithms, circRNA can be systematically detected, thus making them potential diagnostic biomarkers or therapeutic targets. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention provides a lung adenocarcinoma marker, including circ_0006949.

[0006] Preferably, the nucleotide sequence of circ_0006949 is as shown in Seq ID NO:1.

[0007] Preferably, circ_0006949 is a circular RNA.

[0008] Preferably, the marker is a sputum marker.

[0009] Unlike traditional serum biomarkers, sputum samples are closer to lung tissue, allowing for a more accurate reflection of tumor-induced changes. Furthermore, they are less invasive than blood collection and are typically non-invasive. Therefore, to identify specifically upregulated circRNAs in sputum samples from lung adenocarcinoma patients, this invention collected sputum samples from 10 healthy individuals, 10 patients with lung infections, and 30 patients with lung adenocarcinoma. These samples were divided into three groups, and 10 samples from each group were averaged for differential circRNA screening. Differential analysis of the upregulated cirRNAs screened using a cirRNA microarray revealed that 176 circRNAs were specifically upregulated in sputum samples from lung adenocarcinoma patients (see...). Figure 1-2 After analysis, this invention selected hsa_circ_RNA 0006949 (circ_0006949), which may be related to glutamine synthesis and metabolism, for further verification. RNase R experiments confirmed that circ_0006949 is not easily degraded by RNases, confirming it as a circular RNA. Further research collected sputum samples from a large number of healthy individuals and lung adenocarcinoma patients for verification. The results showed that circ_0006949 levels were significantly higher in sputum samples from early-stage lung adenocarcinoma patients than in healthy individuals, indicating its potential as a diagnostic biomarker (see [link to study]). Figure 3 Next, a diagnostic efficacy analysis was conducted between circ_0006949 and common lung cancer serum markers such as CEA, NSE, Cyfra21-1, Carbohydrate antigen 199 (CA199), and pro-gastrin-releasing peptide (ProGRP). The results showed that the area under the curve for circ_0006949 was significantly higher than that of other serum markers. Therefore, its combined use with serum markers can improve the diagnostic efficacy for lung adenocarcinoma (see...). Figure 4 Furthermore, circ_0006949 was significantly downregulated in sputum samples from patients after surgery (see...). Figure 5 Therefore, the circ_RNA discovered in this invention does indeed have the effect of monitoring therapeutic efficacy.

[0010] Preferably, the expression of circ_0006949 is detected in sputum by PCR, with PCR primer sequences as shown in Seq ID NO:2 and Seq ID NO:3.

[0011] The present invention utilizes circ_0006949 as a sputum biomarker for the diagnosis or monitoring of the efficacy of treatment for lung adenocarcinoma, and the steps include the following:

[0012] Step 1: Extract total RNA from sputum. The total RNA extraction procedure follows standard methods in the art. Existing literature-disclosed total RNA extraction techniques or commercially available RNA extraction kits can be applied to this invention, and their specific methods will not be detailed here.

[0013] Step 2: Reverse transcribe the total RNA into complementary deoxyribonucleic acid (cDNA). The reverse transcription step is a conventional method for total RNA extraction in the art. Existing reverse transcription methods or commercially available reverse transcription kits can be used in this invention, and their specific methods will not be described in detail here.

[0014] Step 3: PCR quantitative detection.

[0015] Preferably, in step 3, the upstream and downstream diverter primers for hsa_circ_0006949 in the PCR reaction are the nucleotide sequences shown in Seq ID NO: 2: 5'-ACCATAGTGCACACCTCAGTT-3' and Seq ID NO: 3: 5'-GGGGTATCCATAGCTGTGCT-3', respectively.

[0016] Preferably, in step 3, the upstream and downstream primers for the internal reference gene GAPDH in the PCR reaction are the nucleotide sequences shown in Seq ID NO: 4: 5'-GAGTCAACGGATTTGGTCGT-3' and the nucleotide sequences shown in Seq ID NO: 5: 5'-TTGATTTTGGAGGGATCTCG-3', respectively.

[0017] Preferably, in step 3, the PCR reaction is quantitative real-time PCR; more preferably, the cutoff value for lung adenocarcinoma diagnosis is 1.36 (2). -ΔΔCt >1.36 is positive; 2 -ΔΔCt <1.36 is negative), circ_0006949 2 in sputum -ΔΔCt A value >1.36 is used as the standard for early screening and diagnosis of lung adenocarcinoma; more preferably, 2 -ΔΔCt A value <0.9 is used as the standard for postoperative treatment monitoring in patients with lung adenocarcinoma.

[0018] Preferably, the biomarker circ_0006949 is a lung adenocarcinoma tissue and / or cell biomarker.

[0019] The expression levels of circ_0006949 and its regulated metabolic enzyme gene GLUL (Gene ID: 2752) were higher in lung adenocarcinoma tissues and cells of patients than in normal individuals. circ_0006949 was primarily located in the cytoplasm of both lung adenocarcinoma tissues and cells.

[0020] circ_0006949 is used as a biomarker for lung adenocarcinoma tissues and / or cells. The expression level of circ_0006949 in lung adenocarcinoma tissues and / or cells was detected through the following steps:

[0021] Step 1: Sample collection. Collect lung adenocarcinoma cells, tissue, or adjacent tissue.

[0022] Step 2: Extract total RNA. The total RNA extraction procedure follows conventional methods in the field. Existing literature-disclosed total RNA extraction techniques or commercially available RNA extraction kits can be applied to this invention, and their specific methods will not be detailed here.

[0023] Step 3: Linear RNA elimination. It is preferable to treat the RNA with RNase R. The RNase R used is a commercially available product; refer to the product instructions for specific methods.

[0024] Step 4: The product is reverse transcribed and detected by quantitative PCR.

[0025] Preferably, in step 4, the upstream and downstream primers of GLUL are the nucleotide sequences shown in Seq ID NO: 6: 5'-ctccactgtacggcgtagtc-3' and the nucleotide sequences shown in Seq ID NO: 7: 5'-tgctttccccaacacaccaa-3', respectively.

[0026] Preferably, in step 4, the upstream and downstream diverter primers for hsa_circ_0006949 are the nucleotide sequences shown in Seq ID NO: 2: 5'-ACCATAGTGCACACCTCAGTT-3' and the nucleotide sequence shown in Seq ID NO: 3: 5'-GGGGTATCCATAGCTGTGCT-3', respectively.

[0027] Preferably, in step 4, the upstream and downstream primers for the internal reference gene GAPDH in the PCR reaction are the nucleotide sequences shown in Seq ID NO: 4: 5'-GAGTCAACGGATTTGGTCGT-3' and the nucleotide sequences shown in Seq ID NO: 5: 5'-TTGATTTTGGAGGGATCTCG-3'.

[0028] Preferably, the detection is performed using in situ hybridization or PCR reaction of circ_0006949 to detect one of the following: lung adenocarcinoma tissue, cells, or adjacent tissue.

[0029] Preferably, the circ_0006949 probe and internal control probe selected for in situ hybridization were purchased from Guangzhou Ribo Technology Co., Ltd., with product numbers: lnc1032286 RiboTM h-hsa_circ_0006949_FISH-Probe Mix, lnc110101 RiboTM h-U6 FISH Probe Mix, and lnc110102 RiboTM h-18S FISH Probe Mix. These are commercially available to the public.

[0030] Preferably, the detection is performed using a PCR reaction to detect one of lung adenocarcinoma tissue, cells, or adjacent normal tissue, wherein the circ_0006949 primer of the PCR reaction includes the nucleotide sequences shown in Seq ID NO: 2 and / or Seq ID NO: 3. Preferably, the detection is performed using a PCR reaction to detect one of lung adenocarcinoma tissue, cells, or adjacent normal tissue, wherein the internal control primer of the PCR reaction includes the nucleotide sequences shown in Seq ID NO: 4 and / or Seq ID NO: 5.

[0031] Preferably, the biomarker circ_0006949 is used in combination with at least one of the serum biomarkers Cyfra21-1, CEA, CA19-9, NSE, ProGRP, or SCC. Diagnostic efficacy analysis of circ_0006949 with common lung cancer serum biomarkers Cyfra21-1, CEA, CA19-9, NSE, ProGRP, and SCC revealed that the area under the curve for circ_0006949 was significantly higher than that of other serum biomarkers, indicating that its combined use with serum biomarkers can improve the diagnostic efficacy for lung adenocarcinoma.

[0032] This invention utilizes the circ_0006949 recognition sequence to locate or identify circ_0006949, enabling the diagnosis and treatment monitoring of lung adenocarcinoma. The circ_0006949 recognition sequence includes nucleotide probes and primer sequences that specifically bind to the circ_0006949 sequence; the preferred circ_0006949 sequence is the nucleotide sequence shown in Seq ID NO: 1. The target sites for recognition are sputum, lung adenocarcinoma tissue, cells, or adjacent normal tissue. Sputum sample collection is non-invasive, and the detection is inexpensive, making this method particularly advantageous.

[0033] The present invention also provides the use of the biomarker circ_0006949 described in any of the above claims in the preparation of a lung adenocarcinoma screening kit or a treatment monitoring kit.

[0034] The present invention also provides a kit for screening or monitoring lung adenocarcinoma, the kit detecting any of the biomarkers described above.

[0035] Preferably, the kit includes a circ_0006949 recognition sequence, which is used to identify nucleotide sequences in the target sample.

[0036] Preferably, the circ_0006949 identification sequence includes nucleotide sequences such as Seq ID NO: 2 and Seq ID NO: 3.

[0037] Preferably, the required probes are obtained through commercial purchase, preferably from Guangzhou Ribo Technology Co., Ltd., with product numbers: lnc1032286 RiboTM h-hsa_circ_0006949_FISH-Probe Mix, lnc110101 RiboTM h-U6 FISH Probe Mix, and lnc110102 RiboTM h-18S FISH Probe Mix.

[0038] Preferably, the circ_0006949 recognition sequence is modified with luminescent groups such as fluorescent, biotin, or digoxigenin.

[0039] Preferably, the circ_0006949 recognition sequence is a circ_0006949 primer sequence and / or a circ_0006949 probe sequence.

[0040] Preferably, the circ_0006949 recognition sequence identifies the nucleotide sequence in the target sample, and the recognition method includes PCR reaction, in situ hybridization, etc. The high incidence and mortality rates of lung cancer have made basic and translational research on lung cancer a hot topic. The mortality rate of NSCLC remains high, mainly because the early symptoms of lung cancer are not very obvious, leading to a reduced chance of early diagnosis and treatment. Through high-throughput screening and large-sample validation, we found that circ_0006949 was significantly higher in sputum samples from patients with lung adenocarcinoma than in normal individuals, making it a biomarker for the early diagnosis of lung adenocarcinoma. This finding is original. The sputum sample collection is non-invasive, which is a feature and advantage of this study; moreover, the detection cost is low, making it suitable for widespread application. Attached Figure Description

[0041] Figure 1 In preferred embodiment 1 of the present invention, heatmap analysis is used to identify differentially expressed cirRNAs in sputum samples from normal individuals, patients with lung infections, and patients with lung adenocarcinoma.

[0042] Figure 2 The preferred embodiment of the present invention is shown in the Venn diagram of the differentially expressed cirRNAs in the three groups: LUAD vs. control, infection vs. control, and LUAD vs. infection.

[0043] Figure 3 In preferred embodiment 1 of the present invention, sputum samples were collected from normal individuals and patients with lung adenocarcinoma before treatment, and the expression of circ_0006949 was detected by qRT-PCR.

[0044] Figure 4 The expression of circ_0006949 in sputum specimens of lung adenocarcinoma before surgical treatment and after immunotherapy in preferred embodiment 1 of the present invention.

[0045] Figure 5 In preferred embodiment 1 of the present invention, the diagnostic efficacy of ROC curve analysis of circ_0006949 with commonly used lung cancer serum markers such as Cyfra21-1, CEA, CA19-9, NSE, and ProGRP was evaluated.

[0046] Figure 6The results of the RNase tolerance experiment of H1299 and A549 cell lines in preferred embodiment 2 of the present invention are used to illustrate this invention.

[0047] Figure 7 In a preferred embodiment 2 of the present invention, qRT-PCR was used to detect the expression of circ_0006949 and GLUL in cancerous tissues and adjacent tissues of patients with lung adenocarcinoma.

[0048] Figure 8 The results of FISH detection of circ_0006949 expression in lung adenocarcinoma and adjacent tissues are from preferred embodiment 2 of the present invention.

[0049] Figure 9 In the preferred embodiment of the present invention, FISH detection of circ_0006949 indicates that it is mainly distributed in the cytoplasm. Detailed Implementation

[0050] The present invention will be described more clearly and completely through the following embodiments, but the described examples are only a part of the embodiments of the present invention, and not all of them. The embodiments are provided to help understand the present invention and should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] This embodiment mainly uses the design of a PCR reaction system as an example to illustrate how to detect circRNA expression in the sputum of patients with lung adenocarcinoma, and performs statistical analysis on its diagnostic and therapeutic monitoring value.

[0053] 1. Screening for the expression of circRNAs in LUAD sputum samples

[0054] To identify specifically upregulated circRNAs in LUAD, we collected sputum samples from 10 healthy individuals, 10 patients with pulmonary infection, and 30 LUAD patients, divided into three groups. Ten samples from each group were averaged and screened for differentially expressed circRNAs. circRNA expression profiling was performed using circRNA microarrays at Aksomics (Shanghai, China). Total RNA from each sample was quantified using a NanoDrop ND-1000. Sample preparation and microarray hybridization were performed according to the standard Arraystar protocol. In short, total RNA was digested with RNase R (Epicentre, Inc.) to remove linear RNA and enrich circular RNA. The enriched circular RNA was then amplified and transcribed into fluorescent cRNA using a random primer scheme (Arraystar Super RNA Labeling Kit; Arraystar). The labeled cRNA was hybridized to the Arraystar Human circRNAArray V2 (8x15K, Arraystar). After cleaning the slides, the array was scanned using an Agilent Scanner G2505C. The obtained array images were analyzed using Agilent Feature Extraction Software (version 11.0.1.1). Quantization, normalization, and subsequent data processing were performed using the limma package in R software. Differentially expressed circRNAs between two samples were identified by filtering for folding variations. Hierarchical clustering was performed to reveal different circRNA expression patterns among samples. The circRNA microarray data have been uploaded to the GeneExpression Omnibus (GEO) database (GSE216785).

[0055] 2. Specimen Validation: Sputum specimens from 94 healthy individuals and 146 patients with lung adenocarcinoma (95 of whom underwent treatment before and after lung adenocarcinoma treatment) were provided by Tianjin Medical University Cancer Hospital and divided into two groups. All sputum specimens had clear diagnostic records and were reviewed by the ethics committee.

[0056] 3. Total RNA Extraction: All consumables used in the experiment must be RNase-free. Take 250 μl of sputum sample, add 1 mL of TRIzolLS (Cat#9113, Takara, Japan), homogenize using a homogenizer, add 1 / 5 volume of chloroform, vortex to mix for about 30 seconds, let stand at room temperature for 15 minutes, centrifuge (12,000 rpm, 4℃) for 15 minutes, and carefully aspirate the supernatant. Transfer the collected supernatant to a new Eppendorf tube, add an equal volume of isopropanol, mix by inverting repeatedly, let stand at room temperature for 30 minutes, and centrifuge (12,000 rpm, 4℃) for 30 minutes. Discard the isopropanol, add 1 mL of 75% ethanol prepared using DEPC. Gently mix, centrifuge (12,000 rpm, 4℃) for 5 minutes. Repeat the above steps, discard the 75% ethanol, and air-dry the precipitate. Add approximately 15–25 µL (depending on yield) of DEPC-treated TE buffer or water to the RNA precipitate. Assess the purity and concentration of the RNA solution using a NanoDrop 2000 and store at -80°C for later use. Samples with a total mass greater than 1 μg can be used for further experiments.

[0057] 4. Reverse Transcription: Total RNA was reverse transcribed into complementary deoxyribonucleic acid (cDNA) using the Hifair® III 1st Strand cDNA Synthesis Kit (gDNA digesterplus) (Cat#19332). Following the kit instructions, residual genomic DNA was first removed. The mixture was prepared in an RNase-free centrifuge tube (see Table 1 for the preparation details). The mixture was gently pipetted and incubated at 42°C for 2 min.

[0058] Table 1. Preparation of reverse transcription system mixture

[0059]

[0060] Preparation of reverse transcription reaction system (20 μL system): Add the contents of Table 2 to the reaction tube after removing residual genomic DNA, and gently mix with a pipette.

[0061] Table 2. Preparation of reverse transcription reaction system

[0062]

[0063] The standard reverse transcription procedure is shown in Table 3.

[0064] Table 3. Standard reverse transcription procedure

[0065]

[0066] The reverse transcription product was stored at -20°C for later use.

[0067] 5. Quantitative Real-Time PCR: The expression level of circRNA in the samples was detected using the 2×SYBR Green qRT-PCR Mix (With ROX) kit (Sparkjade). PCR primers were synthesized by Sangon Biotech. The forward and reverse primers for hsa_circ_0006949 were the nucleotide sequences shown in Seq ID NO: 2: 5'-ACCATAGTGCACACCTCAGTT-3' and Seq ID NO: 3: 5'-GGGGTATCCATAGCTGTGCT-3'. The forward and reverse primers for the internal control gene GAPDH were the nucleotide sequences shown in Seq ID NO: 4: 5'-GAGTCAACGGATTTGGTCGT-3' and Seq ID NO: 5: 5'-TTGATTTTGGAGGGATCTCG-3'. The reaction volume (20 μL) was as follows, according to the kit instructions:

[0068] Table 4. Preparation of Real-Time Quantitative PCR System

[0069]

[0070] Perform the following procedure using a real-time quantitative PCR instrument:

[0071] a. Enzyme activation: 95℃, 2 min

[0072] b. Denaturation-annealing-extension: 95℃, 5 seconds;

[0073] 60℃ (adjust according to primer annealing temperature), 30 sec

[0074] 40 cycles

[0075] c. Melting: 95℃, 10s

[0076] 65℃ 5s

[0077] Δ=0.5℃

[0078] 95℃ 5s

[0079] 6. Gene Expression Level Calculation: After the quantitative PCR program is completed, a fluorescence threshold is set. The principle is to set it higher than the sample's fluorescence background value (signal of the first 15 cycles), and to select the initial stage of entering the exponential phase as much as possible, so that the true signal is a fluorescence signal exceeding the threshold, thus obtaining the Ct value of each PCR amplification reaction. If the expression level is too low (Ct exceeds the detection limit of 50), Ct is calculated as 50. The reference gene ΔCt method is used for analysis, with the Ct value of the PCR reaction of the intracellular housekeeping gene GAPDH expression as the relative standard. The formula for calculating the relative expression level of the target gene circRNA is: 2 -ΔΔCt .

[0080] 7. Statistical Analysis: The difference in circ_0006949 expression between sputum samples from normal individuals and sputum samples from lung adenocarcinoma was analyzed using a paired t-test. The cutoff value of circ_0006949 was used as the boundary distinguishing lung adenocarcinoma from normal individuals. The tumor diagnostic value of circ_0006949 was assessed using receiver operating characteristic (ROC) curves: the circ_0006949 score was used as the test variable, and the diagnostic result was used as the state variable. SPSS ROC curves were used for plotting, and the area under the curve was analyzed. A p-value ≤ 0.05 was considered statistically significant.

[0081] 8. Results analysis: circ_0006949 is a potential biomarker for the diagnosis and treatment monitoring of lung adenocarcinoma patients.

[0082] To identify specifically upregulated circRNAs in lung adenocarcinoma, we collected sputum samples from healthy individuals, patients with lung infections, and patients with lung adenocarcinoma for differential circRNA screening. Differential analysis of the upregulated cirRNAs screened by cirRNA microarrays revealed that 176 circRNAs were specifically upregulated in sputum samples from lung adenocarcinoma patients (see...). Figure 1-2 , Figure 1 Heatmap analysis was used to identify differentially expressed cirRNAs in sputum samples from healthy individuals, patients with lung infection, and patients with lung adenocarcinoma. Inclusion criteria were: lung adenocarcinoma (LUAD) vs. control > 7, LUAD vs. infection > 7, and infection vs. control < 0.9. Analysis revealed 176 differentially expressed cirRNAs. Figure 2Venn diagrams show the differentially expressed cirRNAs in the LUAD vs. control, infection vs. control, and LUAD vs. infection groups. After analysis, we selected circ_0006949, which may be related to glutamine synthesis, for further validation. Further validation using sputum samples from a large number of healthy individuals and lung adenocarcinoma patients revealed that circ_0006949 was significantly higher in sputum samples from early-stage lung adenocarcinoma patients than in healthy individuals, indicating its potential as a diagnostic biomarker (see...). Figure 3 Sputum samples were collected from healthy individuals and patients with lung adenocarcinoma before treatment, and the expression of circ_0006949 was detected by qRT-PCR. The cutoff value for lung adenocarcinoma diagnosis was calculated to be 1.36 (2). -ΔΔCt >1.36 is positive; 2 -ΔΔCt <1.36 indicates a negative result), indicating that circ_0006949 2 was present in the sputum specimen. -ΔΔCt A value >1.36 can be used for early screening and diagnosis of lung adenocarcinoma. Furthermore, we conducted a diagnostic efficacy analysis of circ_0006949 with common lung cancer serum markers Cyfra21-1, CEA, CA19-9, NSE, and ProGRP. We found that the area under the curve (AUC) of circ_0006949 was significantly higher than that of other serum markers, indicating that its combined use with serum markers can improve the diagnostic efficacy of lung adenocarcinoma (see [link to analysis]). Figure 4 The expression of circ_0006949 in sputum specimens of lung adenocarcinoma before surgery and after immunotherapy was analyzed. The values ​​were: 1 for Combine (AUC=0.9329), 2 for circ_0006949 (AUC=0.8964), 3 for NSE (AUC=0.7601), 4 for SCC (AUC=0.6407), 5 for cyfra21-1 (AUC=0.5827), 6 for CEA (AUC=0.5353), 7 for ProGRP (AUC=0.5153), and 8 for CA19-9 (AUC=0.5255). Table 5 shows the diagnostic efficacy analysis of diagnostic markers in lung adenocarcinoma patients.

[0083] We also collected sputum samples from lung adenocarcinoma patients before and after surgery to detect whether circ_0006949 had a therapeutic monitoring effect. Comparison of imaging data during sample collection revealed that circ_0006949 was significantly downregulated in sputum samples from lung adenocarcinoma patients after surgery (see...). Figure 5 ROC curve analysis was used to compare the diagnostic efficacy of circ_0006949 with commonly used lung cancer serum markers such as Cyfra21-1, CEA, CA19-9, NSE, and ProGRP. (Student's t-test) indicates that the circ_RNA we discovered does indeed have a therapeutic monitoring effect; circ_0006949 2 in sputum samples -ΔΔCt A value <0.9 can be used for postoperative treatment monitoring in patients with lung adenocarcinoma.

[0084] Table 5. Diagnostic efficacy analysis of diagnostic markers in patients with lung adenocarcinoma

[0085]

[0086] Example 2

[0087] This embodiment mainly uses the design of a PCR reaction system as an example to illustrate how to detect the expression of circ_0006949 and its downstream target protein GLUL in lung adenocarcinoma cells and tissues.

[0088] 1. Sample Collection: A549 and H1299 cells were purchased from ATCC and cultured in RPMI 1640 containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Cells were seeded in 6-well plates when they reached exponential growth phase. Total RNA was extracted after the cells reached 100% confluence.

[0089] 2. Total RNA extraction: Add 0.4 mL of TRIzol to the well plate, pipette to lyse the cells, transfer to an RNase-free EP tube, and add 1 / 5 volume of chloroform. The following steps are as described above.

[0090] 3. Linear RNA elimination: Using the Ribonuclease R (RNase R) kit (Lucigen), 5 μg of total RNA from the experimental group (RNase R treated group) and the control group (no RNase R treated group) was mixed with the following reagents (preparation system shown in Table 6):

[0091] Table 6. Preparation of RNase R system

[0092]

[0093] Mix thoroughly by pipetting, incubate at 37°C for 10 min, then incubate at 85°C for 5 s.

[0094] 4. The product was reverse transcribed and detected by quantitative PCR: PCR primers were synthesized by Sangon Biotech. The forward and reverse primers for GLUL were the nucleotide sequences shown in Seq ID NO: 6: 5'-ctccactgtacggcgtagtc-3' and Seq ID NO: 7: 5'-tgctttccccaacacaccaa-3', respectively. As described above.

[0095] The forward and reverse primers for hsa_circ_0006949 are the nucleotide sequences shown in Seq ID NO: 2: 5'-ACCATAGTGCACACCTCAGTT-3' and Seq ID NO: 3: 5'-GGGGTATCCATAGCTGTGCT-3'. The forward and reverse primers for the internal reference gene GAPDH are the nucleotide sequences shown in Seq ID NO: 4: 5'-GAGTCAACGGATTTGGTCGT-3' and Seq ID NO: 5: 5'-TTGATTTTGGAGGGATCTCG-3'.

[0096] 5. Tissue Sample Collection: Lung adenocarcinoma and adjacent normal tissue specimens from 26 patients with lung adenocarcinoma were provided by Tianjin Medical University Cancer Hospital. All tissue specimens had clear diagnostic records and were reviewed by the ethics committee. 0.1g of tissue was excised from each specimen and placed in an RNase-free EP tube. 1mL of TRIzol was added, along with 4-5 steel beads. The tissue was disrupted using a steel bead shaker. 1 / 5 volume of chloroform was added. The subsequent steps were as described above. The product was reverse transcribed and detected by quantitative PCR, as described above.

[0097] 6. Localization detection (FISH) of circ_0006949 in tissue samples:

[0098] All reagents used are provided in FISH kits or prepared according to methods described in the prior art, including prehybridization solution, hybridization solution, clearing solution, proteinase K, PBS, SSC, DAPI staining solution, and anti-fluorescence quenching mounting medium. Commercially available FISH kits are suitable for this invention.

[0099] a. Dewaxing: Remove the histochemical slides and place them in an oven at 60℃ for 1-4 hours for dewaxing. (The histochemical slides mentioned in step a are prepared from samples of collected lung adenocarcinoma or adjacent normal tissue according to the experimental procedures of FISH in the prior art.)

[0100] b. Dewaxing the sections to water: Place the sections in dewaxing and clearing solution I for 15 min, dewaxing and clearing solution II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, air dry, and then soak in DEPC water.

[0101] c. Digestion: Depending on the tissue fixation time, boil the sections in retrieval solution for 10 minutes and allow them to cool naturally. Then, draw gene circles and, based on the different indices of different tissues, add proteinase K (20ug / ml) and digest at 37°C for 15 minutes. Rinse with pure water and then wash three times with PBS for 5 minutes each time.

[0102] d. Prehybridization: Add prehybridization solution and incubate at 37°C for 1 hour.

[0103] e. Hybridization: Discard the pre-hybridization solution, add hybridization solution containing probe circ_0006949 at a concentration of 1:100, and hybridize overnight at 40°C. Washing after hybridization: Wash away the hybridization solution, wash with 2×SSC at 37°C for 10 min, wash with 1×SSC at 37°C for 2×5 min, and wash with 0.5×SSC at room temperature for 10 min.

[0104] f. DAPI counterstaining of the nucleus: Add DAPI staining solution to the slide, incubate in the dark for 8 minutes, rinse, and then add anti-fluorescence quenching mounting medium to mount the slide.

[0105] g. Photography: The slides were observed and images were acquired under a Nikon upright fluorescence microscope. CY3 is excited by red light at wavelengths of 510-560 nm and emitted at a wavelength of 590 nm, producing red light.

[0106] h. Interpretation of Experimental Results: DAPI-stained cell nuclei appear blue under UV excitation, indicating positive expression as fluorescence of the corresponding fluorophore label. CY3 shows red light. In situ hybridization results show that Circ_0006949 is positive in the cytoplasm of lung adenocarcinoma or adjacent tissues. A few nuclei may be positive in the in situ hybridization results, which is normal. Fluorescence intensity varies depending on the expression level.

[0107] 7. Detection of the localization of circ_0006949 in lung adenocarcinoma cells.

[0108] All reagents used are provided in FISH kits or prepared according to methods described in the prior art. Commercially available FISH kits are suitable for this invention.

[0109] a. Sample collection: As described above, A549 and H1299 cells were cultured and placed in confocal dishes. Before the experiment, the cells were collected after the confluence reached 60%-70% for subsequent FISH experiments.

[0110] b. Cell fixation and permeability:

[0111] Wash cells with 1X PBS for 5 min;

[0112] 4% paraformaldehyde, fixed at room temperature for 10 min;

[0113] Wash cells with 1X PBS for 5 min, repeat 3 times;

[0114] Add 1 mL of pre-cooled permeate to each well and let stand at 4°C for 5 min;

[0115] After discarding the permeabilization solution, wash the cells with 1X PBS for 5 min, 3 times.

[0116] c. Probe detection: Using the Ribo™ Fluorescent In Situ Hybridization kit (Ribo).

[0117] Add 200 μL of prehybridization solution to each well and seal at 37 °C for 30 min;

[0118] During the pre-hybridization process, the hybridization solution was preheated at 37°C.

[0119] Under light-protected conditions, add 2.5 μL of 20 μM circ_0006949 FISH Probe Mix stock solution or internal control FISH Probe Mix stock solution to 100 μL of hybridization solution. The FISH kit was purchased from Guangzhou Ribo Technology Co., Ltd. (product number: C10910 Ribo™ Fluorescent In Situ Hybridization Kit), and the probes used were purchased from Guangzhou Ribo Technology Co., Ltd. (product numbers: lnc1032286 Ribo™ h-hsa_circ_0006949_FISH-Probe Mix, lnc110101 Ribo™ h-U6 FISH Probe Mix, lnc110102 Ribo™ h-18S FISH Probe Mix).

[0120] Discard the prehybridization solution in each well, add 100 μL of probe hybridization solution containing the probe, and hybridize overnight at 37°C in the dark.

[0121] Protect from light, at 42°C, wash each well of cells three times with hybridization wash buffer I for 5 minutes each time to reduce background signal;

[0122] Protect from light, at 42°C, wash cells once with hybridization wash buffer II;

[0123] Protect from light, at 42°C, wash cells once with hybridization wash buffer III;

[0124] Protect from light, wash cells with 1X PBS, and incubate at room temperature for 5 min.

[0125] d. DNA staining:

[0126] Protect from light, stain with 1X DAPI staining solution for 10 min, using enough staining solution to cover all cells in the area to be hybridized;

[0127] Protect from light, wash cells three times with 1X PBS, 5 min each time.

[0128] e. Confocal microscopy detection: Confocal microscopy was used for detection. The maximum excitation wavelength of Cy3 labeling was 555 nm, and the maximum emission wavelength was 570 nm.

[0129] f. Interpretation of experimental results: as described above.

[0130] 8. Results Analysis: circ_0006949 is highly expressed in lung adenocarcinoma patient tissues.

[0131] First, through RNase R experiments, we demonstrated that circ_0006949 is not easily degraded by RNases, and is indeed a circular RNA. Figure 6 RNase tolerance experiments in H1299 and A549 cell lines demonstrated that circ_0006949 is a circular RNA and is not easily degraded by RNase R. Next, we examined its expression in lung adenocarcinoma patient tissues, and found that circ_0006949 and its regulated metabolic enzyme gene GLUL were highly expressed in lung adenocarcinoma patient tissues. Figure 7 The expression of circ_0006949 and GLUL in cancerous and adjacent tissues of lung adenocarcinoma patients was detected by qRT-PCR. This result also suggests that lung adenocarcinoma patients can secrete circ_0006949 into sputum samples. Finally, we examined the localization of circ_0006949 in lung adenocarcinoma tissues and cells. FISH results showed that circ_0006949 was mainly located in the cytoplasm in both lung adenocarcinoma tissues and cells. Figure 8 FISH assay was used to examine the expression of circ_0006949 in lung adenocarcinoma and adjacent normal tissues. The scale bar represents 50 µm, and DAPI indicates nuclear localization. Figure 9 FISH analysis showed that circ_0006949 was mainly distributed in the cytoplasm. The scale bar represents 5 µm. U6 is the nuclear expression control, and 18S is the cytoplasmic expression control. (Student's t-test).

Claims

1. Application of reagents for detecting the expression level of lung adenocarcinoma marker circ_0006949 in the preparation of lung adenocarcinoma screening kits.

2. The application as described in claim 1, characterized in that, The marker is a sputum marker.

3. The application as described in claim 2, characterized in that, The expression of circ_0006949 was detected in sputum by PCR. The PCR primer sequences are shown in Seq ID NO:2 and Seq ID NO:3.

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